Modified Exedins and Uses Thereof

ABSTRACT

Novel exendins with modifications at one or more of following positions: 2, 14, 27 or 28 and polyethylene glycol derivatives thereof are provided. These compounds are useful in treating type 2 diabetes as GLP-1 receptor agonists.

FIELD OF THE INVENTION

The present invention relates to long-lasting exendins andpharmaceutical acceptable salts thereof. To be more particular, thepresent invention relates to pegylated exendins and pharmaceuticalacceptable salts thereof, and preparation method thereof as well astheir uses in preventing and treating type 2 diabetes by regulating theblood glucose level due to the stimulation of the secretion of insulinfrom β-cell induced by the Glucagon-like peptide 1 (GLP-1) receptoracting with said compounds.

BACKGROUND ART

Recently, accompanying with the increased living standard, modernizationof living style and aging of society, incidence of diabetes is also keptincreasing on a yearly basic all over the world, of which the situationis especially obvious in developing countries. Diabetes has become thethird major chronic non-communicable disease next to malignant tumors,cardio-cerebrovascular diseases, and constituted the major causes todeath and disability. As reported in WHO report of 1997 that by thattime there are 135 millions of people suffering from diabetes and 175millions are expected to be reached by 2000. In China, a recent reportshows that incidence of diabetes in the population of age over 20 is3.21%. A preliminary estimation shows that there are at least 20millions of diabetes patients in China nowadays, in which over 95% ofthem are type 2 diabetes patients. From 1987 to 1992, the annual outlayfor direct or indirect uses in diabetes in United State increased from 1billion to 92 billion US Dollars. In China, the outlay for treatment ofdiabetes is also increasing at an incredible speed. According to arelated statistical analysis reported in 1993 that up to 2.2 billionsdollars were spend on the treatment of diabetes at that time, in whichneither the cost for the treatment of diabetes complications, outlay forout-hospital treatments and health care nor indirect loss in socialeconomy were included.

Type 2 diabetes can be controlled by moderation of dietary intake,exercise and regulation of the blood-glucose level with medication.Commonly employed medication includes insulin, sulphonylurea, biguanideas well as Glitazone compounds. These compounds help only in promotingthe blood-glucose level back to the normal level while unable to recoverthe impairments, especially to kidney, cardiovascular system, optical ornervous system caused by diabetes complications. These complications areclosely associated with the increased mortality caused by diabetes. Themajor side effects inherent in the first generation of diabeticmedications include low in blood-glucose level, increase in body-weightand dropsy. The acting mechanisms of these medications maybe different,however, none of them is able to protect the insulin-secreting β-cell,thereby, the in vivo blood glucose metabolism and incretion regulationcannot be maintained in normal condition. In most cases, consecutive useof a single medicine renders it no longer effective, which gives rise tothe application of combined drug treatment. Since diabetes patients takeblood pressure-lowering and cholesterol reducing drugs simultaneouslyduring treatment, the long-term effect of this treatment is not stable.Therefore, development of new medications to cooperate with currentmedications for the regulation of blood glucose level, and to achievethe objects in protecting and recovering the functionality of β-cell aswell as adjusting incretion in response to food intake would result in agreat improvement in diabetic treatment.

Investigation of Glucagon-like peptide-1 (GLP-1) receptor agonist is alikely topic. Investigation and development in this field may open a newchapter in the treatment of type 2 diabetes. Glucagon-like peptide-1 wasfirstly discovered in 1984, which is a kind of intestinal secretionhormones. If type 2 diabetics were injected with this hormone, theirblood glucose level can be adjusted to a normal level (Nathan, D M, etal. Diabetes Care 1992; 15:270-6; Zander, M, et al. Lancet 2002;359:824-30). It was reported that action of Glucagon-like peptide andreceptor agonist thereof is mainly caused by insulin secretion inducedby activating the Glucagon-like peptide 1 receptor on the surface of thepancreas β-cell. Since this effect depends on the in vivo blood glucoselevel, fatal hypoglycemic shock caused by the extremely low bloodglucose level even in the presence of Glucagon-like peptide and receptoragonist thereof would not occur like the traditional medication does.More particularly, when the in vivo blood glucose level is higher than 6mmol/L, GLP-1 remarkably stimulates secretion of insulin, whereas whenthe in vivo blood glucose level reaches the normal level, thestimulation discontinue. Also, this type of agonist stimulates theproliferation of pancreas β-cell of rodent (rat) and also enhances theaction of β-cell tissue. The function that allows the recovery of thepancreas β-cell opens up prospects for the treatment of type 2 diabetesby at least delaying the onset of type 1 diabetes from type 2 diabetes.Meanwhile the Glucagon-like peptide and receptor agonist thereof is ableto inhibit the secretion of glucagon, and thereby make it possible toreduce the output of blood glucose from liver. More importantly, thistype of agonist reduces the dietary intake by inhibiting thegastrointestinal peristalsis and gastric emptying, thereby reduces thebody weight and also helps in controlling the body weight of type 2diabetics.

DETAIL DESCRIPTION OF INVENTION

The objective of the present invention is to provide long-lastingpegylated exendins and pharmaceutical acceptable salts thereof. They caninduce the secretion of insulin and decrease the blood glucose level byactivating Glucagon-like peptide 1 (GLP-1) receptor and thereby usefulin treating and preventing type 2 diabetes. This type of compounds havea long retention time in vivo and exhibits a prolonged action therein.The prolonged retention is not only due to the delay of renal excretioncaused by pegylation, but also due to the improved in vivo enzymatic andchemical stability of the peptide backbone resulted from the pegylation.Pegylation ensures the long-lasting effect of these compounds andthereby reduces the injection times to patients, and patients may getthe benefits of improved quality and effectiveness of such therapy.

More particularly, the present invention relates to, but is not limitedto all pegylated polypeptide precursors listed in the sequence table,and compounds modified with polyethylene glycol with various molecularweights, and pharmaceutical acceptable salts thereof.

Another objective of the present invention is to provide a method forthe preparation of long-lasting pegylated exendins and pharmaceuticalacceptable salts thereof.

Still another objective of the present invention is to provide the useof the long-lasting exendins and/or pharmaceutical acceptable saltsthereof as a Glucagon-like peptide 1 (GLP-1) receptor agonist intreating and preventing type 2 diabetes.

The following technical solutions achieve the objectives of the presentinvention. The present invention relates to exendins and pharmaceuticalacceptable salts thereof whose peptide backbone possesses optimized invivo enzymatic and chemical stability. Particularly, the presentinvention relates to exendins comprising (A) amino acid sequences of SEQID Nos 4 to 141, (B) amino acid sequences substantially identical tothose of SEQ ID Nos. 4 to 141.

The present invention also relates to exendins and pharmaceuticalacceptable salts thereof derived from single or multiple pegylation atposition 2, 14, 27, 28 of the exendins amino acid sequences of SEQ IDNos. 4 to 141, in which molecular weight of said polyethylene glycol iswithin the range of 5,000 to 80,000, preferably 20,000 to 60,000. Theamino acids of the exendins of the present invention possess criticalsites for modification, which include position 2, 14, 27, 28 of theamino acid sequences of exendins.

Also, the objective of the present invention is to provide a method forthe preparation of the above-mentioned exendins and pharmaceuticalacceptable salts thereof, which includes solid-phase and solution-phasesynthesis, purification by reverse-phase high performance liquidchromatography, ion-exchange and gel filtration, and lyophilization.

The present invention further provides the use of the exendins orpegylated exendins and pharmaceutical acceptable salts thereof intreating and/or preventing type 2 diabetes.

Clinic trails show that when type 2 diabetics, whose blood glucose levelwas poorly controlled, were subjected to Glucagon-like peptide 1 (GLP-1)treatment, their fasting blood glucose level become normal (Gutniak, etal., New Eng. J. Med. 326:1316-1322, 1992). Long term administration ofGlucagon-like peptide 1 (GLP-1) can restore the functions of β-cell tonormal level (Rachman, et al., Diabetes 45:1524-1530, 1996).Glucagon-like peptide 1 (GLP-1) can restore the glucose-responsefunction of β-cell in those patients having functional imperfection ofglucose tolerance (Byrne, et al., Diabetes 47:1259-1265, 1998). SinceGlucagon-like peptide 1 (GLP-1) is readily inactivated by dipeptidylpeptidase (DPP IV) in vivo and many cleavage-points for otherendopeptidase (NEP24.11) are present in the Glucagon-like peptide 1(GLP-1), the in vivo lasting time of Glucagon-like peptide 1 (GLP-1) isshort. Promising therapeutic effects of Glucagon-like peptide 1 can beachieved only by means of continuous administration. In this regard,researchers focus on the development of a more stable Glucagon-likepeptide 1 (GLP-1) receptor agonist, mainly formed as modifiedGlucagon-like peptide 1 (GLP-1). More importantly, in the late 1980s andthe early 1990s, Eng et al. isolated Exendin-4 from the saliva secretionorgans of the Gila monster (Heloderma Sespectrum) in southwesternAmerica (Eng, J. et al., J. Biol. Chem., 265:20259-62, 1990, Eng, J., etal. J. Biol. Chem., 267:7402-05, 1992). Exendin-4 is a polypeptidehaving 39 amino acids, which shows 53% homology with Glucagon-likepeptide 1 (GLP-1). Exendin-4 shows affinity to GLP-1 receptor, and itpossesses stronger ability than GLP-1. Its ability in adjusting glucosemetabolism is better than GLP-1; its minimum concentration for thestimulation of insulin secretion is lower than GLP-1; and moreimportantly, the in vivo half-life of Exendin-4 is longer than that ofGLP-1 (Kudsen, L. B. J. Med. Chem. 47:4128-4134, 2004). These are mainlydue to the unique enzymatic stability of Exendin-4, which is originatedfrom the elimination of the cleavage-sites of endopeptidase (such asNEP24.11).

Compounds which possess the function of Glucagon-like peptide 1 (GLP-1)receptor agonist, such as GLP-1 (7-36), GLP-1 (7-37), Exendin-4 andother derivatives of GLP-1 and Exendin-4, have been widely reported inmany publications, which include WO98/43658, WO00/15224, WO00/66629,WO01/98331, WO01/04156, U.S. Pat. No. 5,545,618, U.S. Pat. No. 5,118,WO03/058203, U.S. patent Application Ser. No. 60/395,738, WO04/022004and their references cited therein.

Naturally existing GLP-1 receptor agonists are provided in the followingtable:

SEQ ID Peptide Sequence NO GLP-1 HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2 1(7-36) GLP-1 HAEGTFTSDV SSYLEGQAAK EFIAWLVKGRG 2 (7-37) Exendin-4HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS-NH2 3

Abbreviation in the above sequences: H (His) histidine, A (Ala) alanine,E (Glu) glutamic acid, G (Gly) glycin, T (Thr) threonine, F (Phe)phenylalanine, S (Ser) serine, D (Asp) aspartic acid, V (Val) valine, Y(Tyr) tyrosine, L (Leu) leucine, Q (Gln) glutamine, K (Lys) lysine, I(Ile) isoleucine, R (Arg) arginine, M (Met) methionine, N (Asn)asparagine, P (Pro) proline.

Although more stable GLP-1 receptor agonists have been developed in manylaboratories, their in vivo lasting time is still short, and therebydevelopment of long-lasting derivatives of exendins acting as GLP-1receptor agonists is in great demand. Since the window for thetherapeutic effect and side effect (vomit and nausea) is relativelynarrower, the use of sustained release formulation affords only a smallchance of success. The only possible way to obtain a long-lasting GLP-1receptor agonist is to prepare a stable compound having sufficientlylong in vivo retention time.

Incorporation of polyethylene glycol into active protein or polypeptideincreases the retention time of active protein and polypeptide. Thistechnology has been successfully applied in many protein-basedbiological drugs, such as PEG-Intron, PEGASYS, Neulasta and Somavert andthe like. The methods and chemistry for the incorporation ofpolyethylene glycol into protein and peptide backbone are provided inrelevant references, such as the review by Veronese (Veronese, F M,Biomaterial 2001 22:405-417). In view of the fact that both GLP-1 andExendin-4 belong to GLP-1 receptor, U.S. Pat. No. 5,424,286 and PCTWO98/05351 disclose the comparative experiments of GLP-1 and Exendin-4in terms of their in vivo insulinotropic secretion function. Theexperiments showed that Exendin-4 exhibited a stronger and longer invivo effect than GLP-1 due to its higher stability against the in vivopolypeptide hydrolase (DPP IV, NEP24.11 and the like). PCT WO2004/022004discloses the pegylated GLP-1 receptor agonist, and proposes that whenpolyethylene glycol with molecular weight more than 30,000 daltons isemployed, side effects, such as nausea and vomit, caused by theactivation of the intracephalic GLP-1 receptor, are unlikely to occurwith the resulting derivatives. It indicates that pegylated GLP-1receptor agonist does not only prolong the in vivo acting time, but alsominimize its side effects. However, this type of compounds show noimprovement in the in vivo enzymatic and chemical stability of theirpolypeptide backbone in addition to the limitation in their in vivo orin vitro activity, which limits this type of compound acting as desiredlong-lasting therapeutic agent. The reduced in vivo and in vitroactivity may increase the production cost of long-lasting therapeuticagent. In view of the above reasons, using Exendin-4 backbone as theprecursor in pegylation may afford a greater chance of success inpreparing long-lasting therapeutic agent, in which the polypeptidebackbone possesses better enzymatic stability. Although PCT WO00/66629discloses the resulting compounds and methods involving Exendin-4 as theprecursor in pegylation, there is still a long way to go for asuccessful preparation of a long-lasting therapeutic agent with lowproduction cost. It is because cleavage is likely to occur to His-Glyresidue at the N-terminal by dipeptidyl peptidase (such as DPP IV),which renders the GLP-1 receptor agonist inactive no matter thatExendin-4 is able to prolong the in vivo retention time from a few hoursto several dozen of hours, or even longer. Meanwhile, the long-lastingpegylated GLP-1 receptor agonist should have good chemical stability,especially at the in vivo temperature, i.e., 37° C., which is highlyrequired for Exendin-4, of which the methionine residue at position 14of Exendin-4 backbone is readily undergone oxidation giving rise to themutation of its biological activity, by which preparation of therapeuticagent is made troublesome; and furthermore, hydrolysis of the asparagineresidue at position 28 is the major cause for the inactivation oftherapeutic agent as well as the preparation problem, the mechanism ofhydrolysis is shown as below:

From the mechanism, it shows that hydrolysis of the five-membered ringderived from asparagine does not only decrease the activity of GLP-1receptor agonist, but also cause to the separation of polyethyleneglycol from the polypeptide backbone, and thereby adversely effect thein vivo retention time of the long-lasting compound. Accordingly,modification on glycine at position 2 enhances the enzymatic andchemical stability of the Exendin-4 polypeptide backbone; andmodifications on methionine at position 14 and on asparagine at position28 enhance the chemical stability of Exendin-4 polypeptide backbone aswell. PCT WO00/66629 emphasizes on the preparation of polyethyleneglycol conjugate via acylation with the amino group of the lysine sidechain incorporated during pegylation of Exendin-4. Since Exendin-4itself possesses lysine, selectivity of the acylation reaction is onlyachievable with suitable use of protecting groups, and thus renders theproduction cost higher. By locating the connection point between thepolyethylene glycol for modification and the regiospecific group at thecarboxyl terminal (C-terminal) of the polypeptide, action between thepolypeptide and the receptor would not be affected by the polyethyleneglycol, whereas a regiospecific reaction can be achieved, and therebylowers the production cost.

The present invention discloses a series of derivatives of Exendin-4pegylated at position 2, 14, 27, or 28, as well as the exendins obtainedfrom pegylation conducted with these polypeptide backbones. Thesepegylated exendins exhibit long-lasting effect in vivo, which can beformulated as long-lasting therapeutic agent for injection use.

The exendins of the present invention allows the in vivo and in vitroactivation of the GLP-1 receptor which locates on the surface of β-cell,which further allows the secretion of insulin and thereby lowers theblood glucose level. Examples of the exendins include, but not limitedto, the polypeptide sequences in table 1 as well as those pegylatedcompounds. Serine at position 39, where pegylation takes place, can besubstituted with cysteine or other mercapto-containing synthetic aminoacid. Similarly, multiple pegylations can be achieved in the followingway, in which two or more mercapto-containing amino acids (such ascysteine) are added to the carboxyl terminal, and the resultingelongated polypeptides derivatives may serve as the pegylationprecursor. The general formula for the precursor of two-sitemodification is Cys₍₃₉₎-(Xaa)_(n−1)-Cys_((n+39)), wherein n=0-10, Xaa isany one of the amino acids.

The above-mentioned polypeptides can be prepared by chemical syntheticmethods, which include liquid-phase synthesis of fragment, solid-phasesynthesis (see Merrifield, J. Am. Chem. Soc. 1963, 85:2149-2154), orcombined method of solid-phase and liquid-phase; polypeptide synthesiscan be conducted manually or automatically. Applied Biosystems 431Apolypeptide synthesizer, Csbio polypeptide synthesizer and the like canbe employed in automatic synthesis; and also combinatorial synthesis canbe used in polypeptide synthesis.

Purification by preparative HPLC is required for the polypeptidesprepared by chemical synthetic method, reveres phase materials arecommonly used as the column packing materials (such as C₄, or C₈, orC₁₈). In vivo and in vitro studies of the therapeutic effectiveness areonly allowed after characterizations with analytical identifications(such as high performance liquid chromatography (HPLC), massspectroscopy (MS), amino acids analysis (AAA)). After purification bypreparative HPLC, products can be afford after lyophilization

Polyethylene glycol can be purchased from a variety of suppliers orsynthesized by common methods. Molecular weight of polyethylene glycolis usually within the range of 5,000-80,000 daltons, preferably20,000-60,000 daltons and more preferably about 40,000 daltons.

Polyethylene glycol should be connected with polypeptide at theC-terminal of the polypeptide, so as to minimize the interferencescaused by the polyethylene glycol to the action between polypeptide andthe receptor. That is to say, polyethylene glycol may connect to anyresidues locating between positions 29 to 39, which involvessubstitutions of any one or any few of the amino acids withmercapto-containing amino acid (such as cysteine). In the case of singlepegylation, it is better to substitute serine locating at position 39,carboxyl terminal with cysteine; similarly, in the case of two-sitemodification, the best way is to substitute serine at position 39 withcysteine and add another cysteine at position 40 or 39+n (n=1-10).

The method for bonding to polyethylene glycol via cysteine or mercaptoare widely described in many publications (see Veronese, Biomaterials2001, 22:405-417). People skilled in the art can link polyethyleneglycol with mercapto-containing exendins.

Particularly, bonding via mercapto group can be achieved by way of thefollowing:

1) Mercapto group originates from polypeptide chain. Achieved byincorporating the undermentioned amino acid:

By this time, polyethylene glycol should possess Michael additionacceptor, such as the double bond of maleimide, halogen or sulfonic acidesters substituted groups. Bonding is achieved by forming a thioetherbond between polypeptide and polyethylene glycol.2) Mercapto group originates from the side chain of the amino acid of amodified polypeptide, for example, mercapto group connects with theamino group of the lysine side chain. The amino acid with its side chainmodified in the form of the following formula:

By this time, polyethylene glycol should possess Michael additionacceptor, such as the double bond in maleimide, halogen and sulfonicacid esters substituted groups; bonding is achieved by forming athioether bond between polypeptide and polyethylene glycol.

3) Mercapto group originates from polyethylene glycol. By this time, theconnection point in the polypeptide should contain Michael additionacceptor, such as the double bond in maleimide, halogen and sulfonatesubstituted groups. Bonding is achieved by forming a thioether bondbetween polypeptide and polyethylene glycol.4) If both polyethylene glycol and polypeptide contain Mercapto groups,bonding can be achieved via the formation of asymmetric disulfide bond.

Preferably, covalent bond between polyethylene glycol and polypeptide ofthe present invention is achieved by the formation of a thioether bondin between. However, it is not the only way to link polyethylene glycolwith the polypeptide sequence disclosed in the present invention. Otherconnection methods, such as acylation, reductive amination and oximeformation, are also included in the present invention.

The polypeptide derivatives listed in table 1 are suitable precursorsfor pegylation. However, they are included in the present invention byway of illustration only and the present invention is not limited tothese sequences. In the sequence table, preferred sequences are selectedfrom SEQ ID NO 80 to SEQ ID NO 141.

These pegylated exendins and polypeptide precursors thereof areamphoteric compounds, which can react with acids or bases to form salts.Commonly employed acids for salt formation are selected fromhydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid,phosphoric acid, p-toluenesulfonic acid, methanesulfonic acid, oxalicacid, p-bromobenzene sulfonic acid, carbonic acid, succinic acid, citricacid, benzoic acid, acetic acid, trifluoroacetic acid and the like.Examples of these salts include sulfate, pyrosulfate, hydrosulfate,sulfite, bisulphite, phosphate, hydrophosphate, dihydric phosphate,metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydriodate,acetate, propionate, caprate, caprylate, acrylate, formiate,isobutyrate, caproate, heptylate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, p-methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, r-hydroxybutyrate,glycerate, tartarate, methanesulfonate, propanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and thelike. Preferred acid addition salt is selected from hydrochloride,sulfate, acetate, trifluoroacetate; commonly employed bases for saltformation are selected from sodium hydroxide, potassium hydroxide,ammonia, potassium carbonate and the like.

The exendins of the present invention, particularly the pegylatedexendins, can be used in preventing and treating type 2 diabetes,especially to those patients who present abnormal secretion caused byoverweight or even obesity, due to their potential in recovering β-cell.

Accordingly, the present invention also relates a method for thetreatment and prevention of type 2 diabetes, wherein effective dosage ofthe exendins of the present invention is administered to patients who inneed thereof.

The exendins of the present invention can be used alone, and moresuitably used in combination with other anti-diabetic medicaments (suchas PPAR agonist, sulphonylurea, non-sulphonylurea (Secretagogues),α-glucosidase inhibitor, insulin sensitizer, insulin Secretagogues,glycogen-releasing inhibitor, insulin and other anti-obesitymedicaments) in the treatment of diabetes.

Clinical dosage should be determined according to the actual therapeuticeffectiveness of the various compounds, which is in the range of 0.0001mg/kg to about 200 mg/kg body weight, preferably from 0.001 mg/kg to 20mg/kg body weight, most preferably from 0.01 mg/kg to 1 mg/kg bodyweight. Routes of administration include injection methods (includingintravenous, intramuscular and subcutaneous injection) or othercontinuous injection methods. These compounds can be formulated in avariety of preparations, and administered by conventional routes ofadministration, such as oral and transdermal administration, pulmonary,nasal, buccal spray, suppository administration and the like.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the LC-MS spectrum of SEQ ID No 95.

FIG. 2 shows the influence of PEG-EX-4 analogue on Glucose Tolerance ofdb/db mice on the first day of subcutaneous injection.

FIG. 3 shows the influence of PEG-EX-4 analogue on Glucose Tolerance ofdb/db mice on the third day after subcutaneous injection.

FIG. 4 shows the influence of PEG-EX-4 analogue on Glucose Tolerance ofdb/db mice on the sixth day after subcutaneous injection.

FIG. 5 shows the influence of PEG-EX-4 analogue on Glucose Tolerance ofdb/db mice on the ninth day after subcutaneous injection.

FIG. 6 shows the reduction effect on blood glucose level of mice aftersubcutaneous injection of PEG-EX-4 analogue (1100 μg/kg).

FIG. 7 shows the reduction effect on blood glucose level of mice aftersubcutaneous injection of PEG-EX-4 analogue (3300 μg/kg).

PREFERRED EMBODIMENTS OF THE INVENTION

The examples provided hereinafter assist in better understanding thepresent invention, which are not intended to limit the presentinvention.

Example 1 Solid-Phase Synthesis of Compound SEQ ID No 95 of the PresentInvention (1) Amino Acid Monomers Used in the Synthesis

Fmoc-His(Trt)-OH, Fmoc-dAla-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Nle-OH,Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Arg (pbf)-OH, Fmoc-Ile-OH,Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OHAbbreviation of the above: Fmoc: 9-fluorenylmethoxycarbonyl; Boc:tert-butoxycarbonyl; Trt: trityl; OtBu: t-butoxy; tBu: t-butyl.(2) The Reagents used: N,N-diisopropylethylamine,diisopropylcarbodiimide (DIC), N,N-dimethylformamide (DMF),dichloromethane, hexahydropyridine, 1-hydroxybenzotriazole, Rink amideresin, ninhydrin, methanol, anisole, triisopropylsilane, trifluoroaceticacid.

(3) Experimental Procedure

-   -   A. Synthesis: To 0.5 g (0.25 mmole) Rink amide resin in a        reactor vessel, 1 mmol amino acid was added, activation was        conducted with DIC/HOBT method, and the synthesis was conducted        starting from C-terminal to N-terminal according to the        polypeptide sequence. The reaction was conducted at 25° C. (room        temperature) following the operating procedure below:        -   1. Fmoc group was deprotected by treating with 20%            hexahydropyridine in DMF, 10 min for each time.        -   2. The resins were washed with 10 mL DMF for three times,            and then dried with pump.        -   3. The protected amino acid (1 mmol) and HOBT (1 mmol) were            weighed out and then dissolved in 10 ml DMF followed by            addition of DIC (1 mmol), and then activated for 10 minutes.        -   4. The activated amino acid solution was added to the            reactor vessel and then shaked for 1 hour.        -   5. The resins were washed with DMF for three times, and then            dried with pump.        -   6. Steps 1-5 were repeated for the next cycle in the case of            negative result for the ninhydrin test, whereas steps 3-5            were repeated in the case of positive result for the            ninhydrin test.    -   After the synthesis of polypeptides, the resins were completely        washed with methanol and then dried in air.    -   B. Deprotection of the Protecting Groups and Cleavage of        Polypeptides    -   To 1 g resin having the polypeptide in the reactor vessel was        added the cleavage solution in the following proportion.

Solvents Amount (mL) Anisole 2 Methanol 2 Triisopropylsilane 2Trifluoroacetic Acid 6

-   -   -   The content in reactor vessel was shaked for 2 hours at room            temperature, and then filtered. The filtrate was collected            and the resins were washed with a slight amount of acetic            acid. The collection fluids were combined. After            concentration, ethylether was added and precipitate was            generated. Precipitate was washed with a slight amount of            ethylether to afford the crude product.

    -   C. Purification with High Performance Liquid Chromatography and        Lyophilization

    -   The resulting crude product was dissolved in 10% acetic acid        solution, the solution was injected into the HPLC system for        purification, followed by lyophilization to afford the product.        The resulting polypeptide was analyzed and confirmed as the        desired compound using Chromatography-Mass Spectrometry.

    -   Column: luna C18 (2), 5μ, 100 Å

    -   Detective wavelength: λ=220 nm, Waters preparative system

    -   Gradient: (TFA: trifluoroacetic acid)

T (minute) A: (0.05% TFA) CH₃CN B: (0.05TFA) H₂O 0 10% 90% 20 45% 55% 3045% 55% 30.1 10% 90%

-   -   The molecular weight of the resulting compound: 4212.6 g/mol;        the theoretical molecular weight: 4213 g/mol.

FIG. 1: LC-MS spectrum of SEQ ID No 95

Example 2 Method for Pegylation of Exendins

Pegylation of exendins can be conducted with conventional method.Pegylation of peptides is achieved by modifying mercapto group in theformation of a thioether bond between polyethylene glycol and peptide.To be more particular, one or more cysteines were added to thecarboxyl-terminal of the optimized Exendin-4 derivatives, followed bypegylation conducted by using polyethylene glycol which contains theMaleimide functional group. Thioether bond was formed after Michaeladdition reaction, and thereby polypeptide was covalently bonded withthe polyethylene glycol. In general, the desired polypeptide wasdissolved in 0.1M phosphate buffer solution, followed by addition ofpolyethylene glycol under anaerobic environment. The molar ratio ofpolyethylene glycol to polypeptide was 1:1 and the pH of the reactionwas 6 to 7.5. Oxidation of the mercapto group may be reduced by additionof EDTA to the reaction solution. After two hours, the reaction solutionwas purified with reverse-phase HPLC system. Excess or unreactedpolyethylene glycol was removed by ion-exchange chromatography. Themolecular weight of the resulting product was analyzed and confirmed bymass spectrum. The purity of the product was analyzed with RP-HPLC andGel-chromatography. Taking the modification of SEQ ID NO 95 as anexample, when 43KD PEG was employed in modification, the yield was70-90% (based on polyethylene glycol).

Example 3 Test for the Stability of the Polypeptide

The Exendin-4 derivatives of the present invention possess the optimizedenzymatic and chemical stability. The following method was used todetermine the chemical stability of some polypeptides in the presentinvention.

1 mg of each sample was dissolved in a buffer solution, which contains150 mM sodium chloride and 20 mM phosphate, from which a solution ofconcentration of 4 mg/ml was prepared and its pH is 8.0. The testingsample solutions were placed in a thermostat of 40° C. LC-MS was used todetermine the purity of polypeptide. Correlation between the reductionratio of the main peak area and time reflects the chemical stability ofthe polypeptide.

TABLE 2 Determination of the stability of Exendin-4 based compoundsPurity (%) 0 day 5^(th) day 10^(th) day 15^(th) day Sample 1 98.2 88.081.6 76.2 Sample 2 98 93.1 90.4 88.2 Sample 3 98.9 98.8 98.8 98.8 Sample4 99.7 99.4 99.0 99.3

wherein, sample 1 is Exendin-4 as control, and the sequence is:

His-Gly²-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met¹⁴-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn²⁸-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser³⁹.

Sample 2: 2#Gly in sample 1 was substituted with d-Ala, and 39# wassubstituted with Cys.

Sample 3: 2#Gly in sample 1 was substituted with d-Ala, and 14# Met wassubstituted with Nle, 28#Asn was substituted with Gln.

Sample 4: sample 2 is covalently bonded with PEG40K via Cys at theC-terminal of sample 2; wherein all the C-terminal of samples 1-4 areamide.

Conclusion:

The sequence of sample 1 is a polypeptide sequence that can be isolatedfrom nature. His-Gly residue at the N-terminal is an ideal substrate fordipeptidase and Met that contained in sample 1 readily undergoesoxidization, and also Asn undergoes self-reaction readily, which rendersample 1 unstable. Substitution at position 2, or 14, or 28 greatlyimprove the stability of the peptides; in addition, substitution at allthe 3 positions allows a greater improvement in the stability of thepeptides than substitution of His-Gly with His-dAla alone. Althoughsample 2 is not very stable, it become very stable after bonding withPEG40K (i.e. sample 4), which shows that PEG is useful in enhancing thestability of polypeptide.

Example 4 Formulation

PEG-EX-4 analogue   5 g m-Cresol 0.04 g Iced acetic acid Appropriateamount Iced sodium acetate Appropriate amount Injection waterAppropriate amount 100 ml/100 bottles

Sterile Preparation Experimental Example 1 Oral Glucose Tolerance Testof Polypeptide

Polypeptide of SEQ ID NO 25 was modified with polyethylene glycol ofmolecular weight of about 40000 (sample 5). Oral glucose tolerance testwas then conducted with normal mice and the results were presented inthe following tables:

Table 1. Subcutaneous injections of sample 5 with various dosages wereadministered to normal mice. The influences on the oral glucosetolerance and the area under the curve of the blood glucose level on thefirst day and the third day after subcutaneous injections were given.

(The First Day)

Blood Glucose level (mg/dl) AUC Group 0 min 30 min 60 min 120 min (mg ·h/dl) physiological 129.7 ± 8.1  223.2 ± 33.4 167.7 ± 34.5 98.1 ± 12.1318.8 ± 42.9 saline Sample 5 (20) 116.6 ± 10.8  95.7 ± 9.9***  88.9 ±28.6*** 65.4 ± 5.1*** 176.4 ± 22.0*** Sample 5 (80) 122.2 ± 9.1   92.1 ±12.4***  76.7 ± 8.7*** 64.9 ± 7.5*** 166.6 ± 16.7*** v.s Con ***P <0.001; n = 10

(The Third Day)

Blood glucose level (mg/dl) AUC Group 0 min 30 min 60 min 120 min (mg ·h/dl) physiological 97.3 ± 19.8 193.6 ± 35.3 171.0 ± 46.7 91.8 ± 18.8295.3 ± 54.0 saline Sample 5 (20) 96.2 ± 8.6  172.8 ± 33.2 132.1 ± 12.4*88.3 ± 7.8  253.6 ± 24.4* Sample 5 (80) 89.5 ± 9.3  149.3 ± 32.4** 108.6± 8.8*** 77.1 ± 8.4* 217.0 ± 21.9*** v.s Con, *P < 0.05, **P < 0.01,***P < 0.001; n = 10

Experimental Example 2 Influence of PEG-EXENDIN-4 (PEG-EX-4) Analogue onType 2 Diabetes db/db Mice

1. Testing animals: species, strains: db/db mice, source: Model AnimalCenter of Nanjing University, body weights of mice: 35 g-50 g, male andfemale in half. Numbers of animal: 45, 5-6 mice in each group. Rearingconditions: rearing in SPF grade animal housing, temperature: 22° C.-24°C., humidity: 45%-80%, illumination: 150 Lx-300 Lx, under the 12 h-lightand 12 h-dark cycle condition.

2. Test Method:

Dosage setting up: 5 administration groups: 0.03, 0.1, 0.3, 1 and 3mg/kg; and a blank control group as well; route of administration:subcutaneous injection; volume of administration: 0.05 ml/kg bodyweight.

(1) Influence on Blood Glucose Level of Non-Fasting db/db Mice

According to the non-fasting blood glucose level and body weight ofmice, db/db mice were divided into blank control group and another 5groups to be administered with PEG-EX-4 analogue, 6 mice in each group,and male and female in half. Animals in each group were administeredwith the testing drug and physiological saline, respectively, by asingle subcutaneous injection. Blood glucose level was detected beforeadministration and also 1, 2, 4, 8, 24 hours after administration.Thereafter, the non-fasting blood glucose level was detected every 24hours. The lasting time for the reduction of blood glucose level of thetesting drugs as well as the variation in food intake and body weightsafter administration were observed.

(2) Influence on Blood Glucose Level of Fasting db/db Mice

According to the non-fasting, fasting blood glucose level and bodyweight of mice, db/db mice were divided into blank control group andanother 5 groups to be administered with PEG-EX-4 analogue, 6 mice ineach group, and male and female in half. After fasting for 5 hours,animals in each group were administered with the testing drug andphysiological saline, respectively, by a single subcutaneous injection.Blood glucose level was detected before administration and also 1, 2hours after administration. Thereafter, the non-fasting and fastingblood glucose level was detected every 24 hours. The lasting time forthe reduction of blood glucose level of the testing drug as well as thevariation in food intake and body weights after administration wereobserved.

(3) Influence on Fasting Blood Glucose Level of db/db Mice

According to the fasting blood glucose level and body weight of mice,db/db mice were divided into blank control group and another 5 groups tobe administered with PEG-EX-4 analogue, and 5 mice in each group. Afterfasting for 5 hours, animals in each group were administered with thetesting drug and physiological saline, respectively, by a singlesubcutaneous injection. 2.5 g/kg of glucose was taken orally 15 minutesafter the above administration. After that, blood glucose level wasdetected immediately after taking glucose (0 min) and also 30, 60 and120 minutes after taking glucose. Oral glucose tolerance test wasconducted on the third day, sixth day and ninth day, respectively, afterthe drug administration. The influences of the testing drug on theglucose tolerance of db/db as well as its lasting time and the variationin food intake and body weights after administration were observed.

3. Test Results: the Results for the Influences of Peg-Ex-4 Analogue onBlood Glucose Level of Db/Db Mice were Presented and Summarized in FIGS.2-5 and Tables 1-6.

(1) Influence on Blood Glucose Level of Fasting and Non-Fasting db/dbMice

TABLE 1 Influence of subcutaneous injection of PEG-EX-4 analogue onfasting blood glucose level of db/db mice (mean value ± SD, n = 6)Before Dosage administration After administration (hrs) Group μg/kg 0 12 24 48 Blank — 11.53 ± 5.73 11.77 ± 6.69  10.27 ± 7.16  11.07 ± 4.4610.07 ± 4.66 control PEG-EX-4 0.03 11.28 ± 2.68 8.40 ± 2.02 6.75 ± 2.02 8.58 ± 2.17 10.97 ± 4.09 analogue 0.1 11.08 ± 5.65 6.70 ± 4.35 5.85 ±4.60  9.12 ± 4.84 11.23 ± 5.89 0.3 11.15 ± 3.33  5.13 ± 1.83* 3.78 ±0.73  5.80 ± 2.63*  6.77 ± 2.18 1 11.42 ± 3.74  4.73 ± 1.91* 3.78 ± 0.83 3.93 ± 0.95**  5.03 ± 1.36* 3 11.00 ± 3.66  3.62 ± 1.07*  3.05 ± 0.67* 4.03 ± 1.20**  3.65 ± 0.76** Dosage After administration (hrs) Groupμg/kg 72 96 120 144 168 192 Blank — 11.53 ± 6.33  14.90 ± 6.81  14.32 ±6.61  14.38 ± 5.10 13.53 ± 7.04 13.20 ± 6.27 control PEG-EX-4 0.03 — — —— — — analogue 0.1 — — — — — — 0.3 8.27 ± 2.59 10.60 ± 3.04  11.15 ±4.98  11.70 ± 3.76 12.60 ± 3.84 — 1 7.15 ± 3.10  8.07 ± 2.29*  8.13 ±1.21* 10.75 ± 1.87 11.07 ± 2.65 12.12 ± 1.31 3 5.80 ± 2.19  6.03 ± 1.09* 5.70 ± 2.23*  7.70 ± 2.64*  9.17 ± 2.32 11.43 ± 2.26

TABLE 2 Influence of subcutaneous injections of PEG-EX-4 analogue ondaily non- fasting blood glucose level of db/db mice (mean value ± SD, n= 6) Before administration After administration (hrs) Group Dosage μg/kg0 24 48 72 96 120 144 168 Blank — 14.70 ± 6.87 17.18 ± 4.47 15.22 ± 5.1615.45 ± 6.02 16.13 ± 6.96 15.12 ± 8.05 15.45 ± 5.91 15.25 ± 6.17 controlPEG- 0.03 14.73 ± 5.00 13.42 ± 4.19 12.88 ± 4.50 15.92 ± 5.39 — — — —EX-4 analogue 0.1 14.52 ± 6.01 15.32 ± 6.62 16.22 ± 3.61 — — — — — 0.314.08 ± 2.66 11.35 ± 5.96 11.57 ± 3.07 15.78 ± 3.56 15.17 ± 2.60 14.17 ±4.48 13.53 ± 4.50 13.72 ± 3.89 1 14.30 ± 3.79  7.02 ± 2.49***  9.17 ±4.45 13.73 ± 7.09 13.63 ± 5.48 12.28 ± 4.30 12.50 ± 5.06 12.68 ± 2.73 314.10 ± .86   5.65 ± 1.73***  7.48 ± .15*  9.87 ± 4.74 13.42 ± 4.8911.92 ± 5.10 12.93 ± 3.72 15.27 ± 2.58 (2) Influence on fasting bloodglucose level of db/db mice

TABLE 3 Influences of PEG-EX-4 analogue on the glucose tolerance ofdb/db mice on the first day after subcutaneous injection (mean value ±SD, n = 5). Before Dosage administration After glucose administration(mmol/l) Group μg/kg 0 30 60 120 AUC Blank — 11.66 ± 4.74 22.36 ± 5.7614.84 ± 7.40 12.74 ± 5.10 31.60 ± 11.67 control PEG-EX-4 0.03 11.64 ±4.51 22.28 ± 6.34 13.44 ± 7.47  9.62 ± 8.27 28.94 ± 13.98 analogue 0.111.54 ± 1.80 20.92 ± 2.99 10.66 ± 1.86  6.02 ± 1.25* 24.35 ± 3.51  0.311.18 ± 4.62 18.10 ± 1.67  9.06 ± 2.23  5.34 ± 1.34* 21.31 ± 4.01  111.54 ± 2.50 16.82 ± 2.38  9.12 ± 4.60  5.26 ± 2.54* 20.77 ± 6.28  311.18 ± 4.37 16.54 ± 4.40  9.10 ± 3.21  4.44 ± 1.74** 20.11 ± 5.98 

TABLE 4 Influence of PEG-EX-4 analogue on glucose tolerance of db/dbmice on the third day after subcutaneous injection (mean value ± SD, n =5) Dosage Before After glucose administration (mmol/l) Group μg/kgadministration 0 30 60 120 AUC Blank — 13.34 ± 6.85  22.28 ± 5.59 18.16± 6.55 14.06 ± 4.94 35.13 ± 11.22 control PEG-EX-4 0.03 11.50 ± 4.75 21.40 ± 4.06 18.64 ± 5.97 13.94 ± 6.10 34.53 ± 10.64 analogue 0.1 10.38± 3.65  19.66 ± 7.27 18.06 ± 2.45 11.72 ± 4.58 31.83 ± 8.03  0.3 7.72 ±2.77 19.52 ± 2.40 16.24 ± 5.68 12.16 ± 5.76 29.95 ± 8.80  1  5.88 ±0.92* 20.18 ± 2.82  8.50 ± 2.88*  7.04 ± 1.71* 21.46 ± 4.02* 3  5.50 ±2.29* 18.24 ± 5.05  9.74 ± 5.57  7.72 ± 4.98 21.66 ± 9.51 

TABLE 5 Influence of PEG-EX-4 analogue on glucose tolerance of db/dbmice on the sixth day after subcutaneous injection (mean value ± SD, n =5) Dosage Before After glucose administration (mmol/l) Group μg/kgadministration 0 30 60 120 AUC Blank — 14.20 ± 6.56 22.96 ± 2.86 18.70 ±7.15 13.70 ± 7.12 35.91 ± 11.33 control PEG-EX-4 0.03 12.62 ± 7.38 22.28± 4.45 17.62 ± 5.40 11.50 ± 6.38 33.26 ± 10.59 analogue 0.1 14.60 ± 3.4925.62 ± 2.45 19.76 ± 2.56 14.12 ± 2.05 38.34 ± 4.32  0.3 11.50 ± 4.5523.58 ± 1.89 18.94 ± 3.86 12.42 ± 4.99 35.08 ± 7.04  1  8.12 ± 1.2226.34 ± 2.09 16.54 ± 3.65  9.68 ± 2.63 32.45 ± 4.92  3  5.80 ± .48*23.66 ± 4.50 11.66 ± 4.37  7.28 ± 2.40 25.67 ± 6.94 

TABLE 6 Influence of PEG-EX-4 analogue on glucose tolerance of db/dbmice on the ninth day after subcutaneous injection (mean value ± SD, n =5) Dosage Before After glucose administration (mmol/l) Group μg/kgadministration 0 30 60 120 AUC Blank — 12.04 ± 8.47  25.90 ± 4.16 18.52± 8.29 14.04 ± 7.91 36.87 ± 14.14 control PEG-EX-4 1 9.60 ± 1.16 24.86 ±1.67 17.90 ± 2.92 12.28 ± 4.08 34.40 ± 4.89  analogue 3 9.36 ± 3.6623.46 ± 2.41 15.60 ± .02  11.84 ± 4.35 31.69 ± 6.64  *P < 0.05; **P <0.01; ***P < 0.001, in comparison with blank control group

Experimental Example 3 Preliminary Testing Results of the Influence ofPeg-Exendin-4 (Peg-Ex-4) Analogue on Blood Glucose Level of KKAy Mice 1.Test Methods:

Single subcutaneous injections of PEG-EX-4 analogue at various dosageswere administered to normal mice. Variation in blood glucose level atdifferent times after injection was detected.

2. Test Results:

(1) See FIG. 6, the reduced blood glucose level of KKay mice lasts for3-4 days after subcutaneous injection of PEG-EX-4 analogue (1100 g/kg).(2) See FIG. 7, the reduced blood glucose level of KKay mice lasts for3-4 days after subcutaneous injection of PEG-EX-4 analogue (3300 g/kg).

TABLE 7 The amino acid sequences of the said long-lasting exendins ofthe present invention were given. Series SEQ ID Number Sequences NO HR1HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS 4 HR2 HGEGTFTSDL SKQMEEEAVRLFIEWLKNGG PSSGAPPPC 5 HR3 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC 6HR4 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhC 7 HR5 HGEGTFTSDLSKQMEEEAVR LFIEWLKNGG PSSGAPPPhChC 8 HR6 HGEGTFTSDL SKQMEEEAVRLFIEWLKNGG PSSGAPPPK* 9 HR7 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPK*K* 10 HR8 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPC-NH₂ 11HR9 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC-NH₂ 12 HR10 HGEGTFTSDLSKQMEEEAVR LFIEWLKNGG PSSGAPPPhC-NH₂ 13 HR11 HGEGTFTSDL SKQMEEEAVRLFIEWLKNGG PSSGAPPPhChC-NH₂ 14 HR12 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPK*-NH₂ 15 HR13 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK*K*-NH₂16 HR14 HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS 17 HR15 HdAEGTFTSDLSKQMEEEAVR LFIEWLKNGG PSSGAPPPC 18 HR16 HdAEGTFTSDL SKQMEEEAVRLFIEWLKNGG PSSGAPPPCC 19 HR17 HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPhC 20 HR18 HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhChC 21HR19 HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPK* 22 HR20 HdAEGTFTSDLSKQMEEEAVR LFIEWLKNGG PSSGAPPPK*K* 23 HR21 HdAEGTFTSDL SKQMEEEAVRLFIEWLKNGG PSSGAPPPS-NH₂ 24 HR22 HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPC-NH₂ 25 HR23 HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPCC-NH₂26 HR24 HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhC-NH₂ 27 HR25HdAEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPhChC-NH₂ 28 HR26 HdAEGTFTSDLSKQMEEEAVR LFIEWLKNGG PSSGAPPPK*-NH₂ 29 HR27 HdAEGTFTSDL SKQMEEEAVRLFIEWLKNGG PSSGAPPPK*K*-NH₂ 30 HR28 HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPS 31 HR29 HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPC 32 HR30HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPCC 33 HR31 HGEGTFTSDLSKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC 34 HR32 HGEGTFTSDL SKQNleEEEAVRLFIEWLKNGG PSSGAPPPhChC 35 HR33 HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPK* 36 HR34 HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPK*K* 37HR35 HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPS-NH₂ 38 HR36 HGEGTFTSDLSKQNleEEEAVR LFIEWLKNGG PSSGAPPPC-NH₂ 39 HR37 HGEGTFTSDL SKQNleEEEAVRLFIEWLKNGG PSSGAPPPCC-NH₂ 40 HR38 HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPhC-NH₂ 41 HR39 HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPhChC-NH₂ 42 HR4O HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPK*-NH₂ 43 HR41 HGEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPK*K*-NH₂ 44 HR42 HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPS 45HR43 HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPC 46 HR44 HGEGTFTSDLSKQMEEEAVR LFIEWLKQGG PSSGAPPPCC 47 HR45 HGEGTFTSDL SKQMEEEAVRLFIEWLKQGG PSSGAPPPhC 48 HR46 HGEGTFTSDL SKQMEEEAVR LFIEWLKQGGPSSGAPPhChC 49 HR47 HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK* 50 HR48HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK*K* 51 HR49 HGEGTFTSDLSKQMEEEAVR LFIEWLKQGG PSSGAPPPS-NH₂ 52 HR50 HGEGTFTSDL SKQMEEEAVRLFIEWLKQGG PSSGAPPPC-NH₂ 53 HR51 HGEGTFTSDL SKQMEEEAVR LFIEWLKQGGPSSGAPPPCC-NH₂ 54 HR52 HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPhC-NH₂55 HR53 HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPhChC-NH₂ 56 HR54HGEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK*-NH₂ 57 HR55 HGEGTFTSDLSKQMEEEAVR LFIEWLKQGG PSSGAPPPK*K*-NH₂ 58 HR56 HdAEGTFTSDL SKQNleEEEAVRLFIEWLKNGG PSSGAPPPS 59 HR57 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPC 60 HR58 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPCC 61 HR59HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC 62 HR60 HdAEGTFTSDLSKQNleEEEAVR LFIEWLKNGG PSSGAPPPhChC 63 HR61 HdAEGTFTSDL SKQNleEEEAVRLFIEWLKNGG PSSGAPPPK* 64 HR62 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPK*K* 65 HR63 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPS-NH₂66 HR64 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPC-NH₂ 67 HR65HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGG PSSGAPPPCC-NH₂ 68 HR66 HdAEGTFTSDLSKQNleEEEAVR LFIEWLKNGG PSSGAPPPhC-NH₂ 69 HR67 HdAEGTFTSDL SKQNleEEEAVRLFIEWLKNGG PSSGAPPPhChC-NH₂ 70 HR68 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPK*-NH₂ 71 HR69 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKNGGPSSGAPPPK*K*-NH₂ 72 HR70 HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPS 73HR71 HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPC 74 HR72 HdAEGTFTSDLSKQMEEEAVR LFIEWLKQGG PSSGAPPPCC 75 HR73 HdAEGTFTSDL SKQMEEEAVRLFIEWLKQGG PSSGAPPPhC 76 HR74 HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGGPSSGAPPPhChC 77 HR75 HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK* 78HR76 HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK*K* 79 HR77 HdAEGTFTSDLSKQMEEEAVR LFIEWLKQGG PSSGAPPPS-NH₂ 80 HR78 HdAEGTFTSDL SKQMEEEAVRLFIEWLKQGG PSSGAPPPC-NH₂ 81 HR79 HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGGPSSGAPPPCC-NH₂ 82 HR80 HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPhC-NH₂83 HR81 HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPhChC-NH₂ 84 HR82HdAEGTFTSDL SKQMEEEAVR LFIEWLKQGG PSSGAPPPK*-NH₂ 85 HR83 HdAEGTFTSDLSKQMEEEAVR LFIEWLKQGG PSSGAPPPK*K*-NH₂ 86 HR84 HdAEGTFTSDL SKQNleEEEAVRLFIEWLKQGG PSSGAPPPS 87 HR85 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGGPSSGAPPPC 88 HR86 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPCC 89 HR87HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPhC 90 HR88 HdAEGTFTSDLSKQNleEEEAVR LFIEWLKQGG PSSGAPPPhChC 91 HR89 HdAEGTFTSDL SKQNleEEEAVRLFIEWLKQGG PSSGAPPPK* 92 HR90 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGGPSSGAPPPK*K* 93 HR91 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPS-NH₂94 HR92 HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPC-NH₂ 95 HR93HdAEGTFTSDL SKQNleEEEAVR LFIEWLKQGG PSSGAPPPCC-NH₂ 96 HR94 HdAEGTFTSDLSKQNleEEEAVR LFIEWLKQGG PSSGAPPPhChC-NH₂ 97 HR95 HdAEGTFTSDLSKQNleEEEAVR LFIEWLKQGG PSSGAPPPK*-NH₂ 98 HR96 HdAEGTFTSDL SKQNleEEEAVRLFIEWLKQGG PSSGAPPPK*K*-NH₂ 99 HR97 HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGGPSSGAPPPS 100 HR98 HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPC 101HR99 HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPCC 102 HR100HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPhC 103 HR101 HdAEGTFTSDLSKQNleEEEAVR LFIEWLQKGG PSSGAPPPhChC 104 HR102 HdAEGTFTSDL SKQNleEEEAVRLFIEWLQKGG PSSGAPPPK* 105 HR103 HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGGPSSGAPPPK*K* 106 HR104 HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPS-NH₂107 HR105 HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPC-NH₂ 108 HR106HdAEGTFTSDL SKQNleEEEAVR LFIEWLQKGG PSSGAPPPCC-NH₂ 109 HR107 HdAEGTFTSDLSKQNleEEEAVR LFIEWLQKGG PSSGAPPPhC-NH₂ 110 HR108 HdAEGTFTSDLSKQNleEEEAVR LFIEWLQKGG PSSGAPPPhChC-NH₂ 111 HR109 HdAEGTFTSDLSKQNleEEEAVR LFIEWLQKGG PSSGAPPPK*-NH₂ 112 HR110 HdAEGTFTSDLSKQNleEEEAVR LFIEWLQKGG PSSGAPPPK*K*-NH₂ 113 HR111 HdAEGTFTSDLSKQMEEEAVR LFIEWLVKGG PSSGAPPPS 114 HR112 HdAEGTFTSDL SKQMEEEAVRLFIEWLVKGG PSSGAPPPC 115 HR113 HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGGPSSGAPPPCC 116 HR114 HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPhC 117HR115 HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPhChC 118 HR116HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPK* 119 HR117 HdAEGTFTSDLSKQMEEEAVR LFIEWLVKGG PSSGAPPPK*K* 120 HR118 HdAEGTFTSDL SKQMEEEAVRLFIEWLVKGG PSSGAPPPS-NH₂ 121 HR119 HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGGPSSGAPPPC-NH₂ 122 HR120 HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPCC-NH₂123 HR121 HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPhC-NH₂ 124 HR122HdAEGTFTSDL SKQMEEEAVR LFIEWLVKGG PSSGAPPPhChC-NH₂ 125 HR123 HdAEGTFTSDLSKQMEEEAVR LFIEWLVKGG PSSGAPPPK*-NH₂ 126 HR124 HdAEGTFTSDL SKQMEEEAVRLFIEWLVKGG PSSGAPPPK*K*-NH₂ 127 HR125 HdAEGTFTSDL SKQNleEEEAVRLFIEWLVKGG PSSGAPPPS 128 HR126 HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPC 129 HR127 HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPCC 130HR128 HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPhC 131 HR129HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGG PSSGAPPPhChC 132 HR130 HdAEGTFTSDLSKQNleEEEAVR LFIEWLVKGG PSSGAPPPK* 133 HR131 HdAEGTFTSDL SKQNleEEEAVRLFIEWLVKGG PSSGAPPPK*K* 134 HR132 HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPS-NH₂ 135 HR133 HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPC-NH₂ 136 HR134 HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPCC-NH₂ 137 HR135 HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPhC-NH₂ 138 HR136 HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPhChC-NH₂ 139 HR137 HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPK*-NH₂ 140 HR138 HdAEGTFTSDL SKQNleEEEAVR LFIEWLVKGGPSSGAPPPK*K*-NH₂ 141In table 7, C, hC, K* are the modification sites for pegylation. C iscysteine, hC is homocysteine and K* is lysine with a modifiedside-chain, such as the mercaptopropionic acid on the amino group of theside chain of lysine. CC, hChC or K*K* in the sequence represent twomodification sites for pegylation. Nle is norleucine, dAlea isD-alanine, —NH₂ is an amide at the C-terminal.

1. Exendins comprising (A) amino acid sequences of SEQ ID Nos. 4 to 141,(B) amino acid sequences substantially identical to those of SEQ ID No 4to
 141. 2. Exendins according to claim 1, comprising exendins obtainedfrom pegylation of amino acid sequences of SEQ ID Nos 4 to 141, andpharmaceutical acceptable salts thereof.
 3. Exendins according to claim2, further comprising exendins obtained from single or multiplepegylations at position 2, 14, 27, 28 of the amino acid sequences of SEQID Nos 4 to 141, and pharmaceutical acceptable salts thereof. 4.Exendins according to claim 2, wherein molecular weight of thepolyethylene glycol is within the range of 5,000 to 80,000.
 5. Exendinsaccording to claim 2, wherein molecular weight of the polyethyleneglycol is within the range of 20,000 to 60,000.
 6. Exendins according toclaim 1, comprising pharmaceutical acceptable salts of the exendins. 7.A method for the preparation of the exendins and pharmaceuticalacceptable salts thereof of claim 1, comprising solid-phase andliquid-phase synthesis, reverse-phase high performance liquidchromatography, ion-exchange and gel filtration for purification as wellas lyophilization.
 8. (canceled)
 9. A method for treatment and/orprevention of type 2 diabetes, comprising administration of effectivedosage of the exendins according to claim 1 to a patient in needthereof.
 10. A method for the preparation of the exendins andpharmaceutical acceptable salts thereof of claim 2, comprisingsolid-phase and liquid-phase synthesis, reverse-phase high performanceliquid chromatography, ion-exchange and gel filtration for purificationas well as lyophilization.